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Gel electrophoresis and flow cytometry are among of the most important tools of biotechnology for the assessment of DNA and protein in the cell. These important methodologies have been used in molecular biology laboratories for several decades to assess the molecular weight and structural parameters of these macromolecules. In the past several years, these molecular biology technologies have become increasingly important as diagnostic tools in hospital based clinical laboratories for the identification of diseases, to assess molecular mechanisms of pathogenesis and to make treatment decisions. This paper is a review of the principles of each of these technologies and their use in clinical and molecular medicine.
Analytical flow cytometry (AFC) is a powerful tool for assessing cell composition based on the assessment of optical properties of cells scanned at the rate of approximately 100 cells per second (Boddy et al 2001; Givan 2001). Among the advantages of this technology are rapid screening and the capacity to make quantitative measurements of individual cells (Davey & Kell 1996; Givan 2001). The technique has been applied to studies in medical microbiology to identify specific strains of bacteria associated with patient infections (Boddy et al 2001). It can also be used to assess the effects of antibiotics on microbial strains to detect sensitivity and resistance parameters rapidly to aid the design and implementation of therapy protocols in patients with acute infections Davey & Kell 1996). The technique can be used to measure the DNA content within a cell, protein content and the activity of specific proteins within a cell (Roederer 2001).
Flow cytometry is a highly sensitive technique that involves measurements of fluorescence that are used to identify and quantify biochemical properties of individual cells (Shapiro 2003). The method involves the detection of optical excitation parameters of fluorescent probes applied to specific cellular components (Shapiro 2003). Optical excitation patterns are standardized by controlled flow using hydrodynamic focusing (Shapiro 2003). The application of multi-beam analysis such as two beam, two channel detection and tom photon excitation systems that can be used to detect two fluorescent signals simultaneously and a high signal to noise ratio (Zhong et al, 2005). Other applications include the use of labeled nanoparticles to target specific cell components for assessment (Zhong et al 2005). Analytical flow cytometry can be used to assess cycle cycle parameters in dividing cells on an individual basis (Pozaroski & Darzynkiewicz 2004). This may involve DNA content assessment using a fluorescent dye such as propidium iodide (Shapiro 2003) In addition bivariate analysis involving the simultaneous assessment of DNA content and proteins involved in cell cycle regulation can be used to decipher the details of this process in normal cells and malignant cells (Pozaroski & Darzynkiewicz 2004).
Cell viability is another measurement that can be made using flow cytometry (Bertho et al 2000). Moreover, the technique can be used to discriminate between cell death mechanisms associated with apoptosis from necrosis (Bertho et al 2000). Cell viability measurements have important clinical applications in pathogenesis to assist in the elucidation of the tissue-destroying effects of disease. For example, the fragmentation of DNA that occurs during apoptosis causes a change in the scatter properties of the molecule (Bertho et al 2000). Likewise, changes in membrane permeabilization associated with necrosis can also be detected using the methods of flow cytometry (Bertho et al 2000). This tool can be very effective in determining the extent of disease as well as the therapeutic effects of treatment protocols in patients (Galanzha et al 2008).
Another important application of flow cytometry to the study of physiology involves its use in the detection of protein: protein interactions. By means of fluorescence resonance energy transfer (FRET) to detect the interactions of proteins that are each tagged with a different fluorescent marker (He et al 2003; Oswald 2004)). This technology can be used to detect homotypic and heterotypic protein interactions that may be important in identifying cell pathways important in specific disease processes (He et al 2003; Maecker & Trotter 2006).
Highly specialized applications of flow cytometry have been utilized to study specific diseases. For example, it is a favored diagnostic tool for patients with Glanzmannââ‚¬â„¢s thrombasthenia, in which the technique can be used to make a differential diagnosis of patient auto-antibodies that result in the destruction of blood platelets Giannini et al 2008). Another application of flow cytometry to clinical medicine has involved the identification of patients with heparin-induced thrombocytopenia, by means of the highly sensitive detection of anti-heparin:P4 antibodies that attack and destroy blood platelets (Gobbi et al 2004).
One of the more recent applications of flow cytometry to molecular medicine involves its use in the assessment of gene silencing mechanisms by small microRNAs (Martinex-Ferrandis et al 2007). Inappropriate gene silencing has been linked to several important disease mechanisms include human malignant disease progression in several types of cancer (He et al 2003). Moreover the use of small interfering RNAs represents a novel therapeutic approach to the treatment of human diseases associated with the production of mutant proteins that cause tissue destruction (Martinex-Ferrandis et al 2007). Flow cytometry assessments involving the use of fluorescently labeled short interfering RNAs (siRNAs) have been used in cell sorting experiments to detect cellular responses to these RNAs with potential therapeutic application (Maeker & Trotter, 2006). This sensitive assessment facilitates the identification of cells in which the siRNA has effectively produced a gene silencing effect (Martinex-Ferrandis et al 2007; Novo & Wood 2008).
Gel electrophoresis is a method of separating DNA and protein macromolecules based on differences in molecular weight (Voytas 2001). The basic format of this technology involves the use of an electric field to achieve the separation of negatively charged molecules as they diffuse a gel polymer (Voytas 2001). The rate of migration of the macromolecule through the gel is determined by the diameter of the pores or openings within the polymerized gel matrix (Voytas 2001). Due to its uniform negative charges provided by the phosphate groups that comprise the sugar phosphate backbone of DNA, the DNA will migrate towards the positively charged electrode when placed in an electric field. Moreover, the rate of migration of linear DNA through the pore matrix of the gel polymer, will be determined directly by the length of the molecule or the number of base pairs in the double stranded helix since the molecule has a fixed diameter (Voytas 2001). The migration pattern of linear DNA can be used to determine the molecular weight of different molecules when compared to the migration rate of DNA standards of known molecular weight, estimated as the number of base pairs (Voytas 2001). The gel polymers used in DNA electrophoresis are agarose, which is used to analyze DNAs of molecular weights ranging from approximately 200-25,999 base pairs (bps), and polyacrylamide, which is used to separate low molecular weight DNAs ranging in size from 1-1000 bps (Voytas 2001).
There are many important applications of DNA electrophoresis in clinical medicine (VanHeukelum & Bartema 2003). DNA mutations that are associated with specific diseases can be identified to provide an important diagnostic tool (VanHeukelum & Bartema 2003). In addition DNA assessment of disease pathogens can provide important information on the etiologic agents associated with infectious disease (Sellers et al 2007). Some applications of this technology involve the testing of DNA from cancer patients as a tool for assessing the type of cancer and its prognosis (Tse et al 2006). For example, different mutations are associated with different types of breast cancer (Tse et al 2006). Inherited forms of the disease are associated with nmutations in the BRCA-1 and BRCA-2 genes, which is important in prognosis, disease recurrence and treatment decisions (Zustin et al 2009). In the area of infectious disease, the identification of specific viruses associated with flu outbreaks is assisted by the analysis of the viral DNA composition (Sellers et al 2007). The comet assay is an application of DNA gel electrophoresis that allows the identification of DNA damage in a single cell (Shapohnikov, 2008). The cells to be assayed are lysed with detergent and embedded in an agarose gel. These embedded cells called nucleoids, migrate to the anode in a comet shaped array whose tail intensity is proportional to the number of double strand breaks in the cellular DNA (Shaposhnikov 2008).
Protein gel electrophoresis also requires a gel polymer and an electric field to separate proteins with different physical and biochemical properties (Carrrette et al 2006). The most common type of protein gel electrophoresis involves the use of denaturing gels, in which a protein is assessed in its denatured or unfolded configuration (Daszykowski et al 2009). Protein denaturation is achieved with the use of detergents and chemicals that break the chemical bonds that hold a protein in its native three- dimensional configuration (Daszykowski et al 2009). The denatured protein is then coated with a solvent, usually sodium dodecyl sulfate (SDS) which provides a uniform negative charged on the natured protein to facilitate separation of different protein molecules based on differences in molecular weight (Daszykowski et al 2009). This method is used to determine the molecular weight of proteins and can be used to compare the same protein in normal and diseases states (Kaczmarek et al 2004). Protein gel electrophoresis can also be carried out on native proteins in their 3D configuration to assess migration pattern difference based on amino acid substitutions and abnormal conformational properties (Carrette et al 2006).
New developments in gel electrophoresis technology have resulted in more sophisticated disease assessment tools in the hospital laboratory. Difference gel electrophoresis (DIGE) is a specialized application of standard gel electrophoresis that corrects for the intrinsic variations in biomolecule electrophoresis migration patterns produced by standard methods of gel electrophoresis (Unlu et al 1997; Minden et al 2009). Standard methods of 2D protein electrophoresis in polyacrylamide gels involve the separation of hundreds of cellular proteins simultaneously in a single gel which produces unavoidable disturbances in electrophoretic mobility of individual proteins (Dowsey et al 2008). This inherent variation in protein migration pattern makes it very difficult to assess reliably the results of different gel patterns. This type of comparative assessment is essential in clinical hospital analyses of proteins associated with disease states in order to evaluate the physiological abnormalities of protein structure and function that may play an important role in disease processes (Dowsey et al 2008). This problem was resolved by the invention of a technology that permitted the simultaneous assessment of proteins from multiple cellular sources on the same gel (Minden et al 2009). This is accomplished by the use of matching sets of fluorescent dyes that facilitates a direct comparison of proteins from different sources (Minden et al 2009). In this way, for example, proteins from cancer cells can be directly compared with the same protein from a normal cell to assess whether changes in the molecular weight or amino acid sequence may correlate with the disease state of the tumor tissue (Minden et al 2009). Additional modifications that have added to the reliability of this procedure are the inclusion of pooled internal protein standard controls and the use of fractionated cell samples as the starting material for protein extraction prior to electrophoretic separation (Daszykowski et al 2009).
Discontinuous native protein gel electrophoresis is a method developed to assess the 3D structural configuration of a protein by gel electrophoresis (Niepmann & Zhang 2006). This method represents an application of standard non-denaturing protein gels that assess the migratory behavior of proteins through polyacrylamide gels (Niepmann & Zhang 2006). Standard gels of his type often cannot be used to assess oligomeric proteins containing multiple subunits such as hemoglobin which is an alpha2-beta2 tetramer. The discontinuous method of protein gel electrophoresis permits the separation and resolution of proteins based on differences in molecular weight, shape conformation and oligomeric state (Rosell et al 2009). This is accomplished by the use of Serva blue G to add negative charge to the proteins using a discontinuous buffer and a gradient gel (Raymer & Smith 2007). The amino acid histidine is used instead of glycine in the gel running buffer to slow the migration rate of basic proteins (Raymer & Smith 2007). The gradient gel facilitates the differential separation of proteins based on conformation (Sellers et al 2007; Tse et al 2009). Taken together, these modifications facilitate the structural assessment of proteins from patients whose diseases are the results of protein abnormalities, such as hemoglobin disorders (ScarontastnÃÂ¡ & Scaronlais 2005).
Flow cytometry and gel electrophoresis are standard technologies used in molecular biology research that have increasingly been implemented in hospital laboratories in the study of human disease. These technologies provide sensitive tools for the analysis of DNA and protein macromolecules that play a role in the etiology of disease pathogenesis. There are many broad-spectrum applications of flow cytometry and gel electrophoresis in the clinical setting, ranging from the identification of microbial pathogens, the identification of abnormal cellular proteins and the assessment of defective genes that may play an important role in disease mechanisms. It is expected that the clinical applications of these technologies to clinical medicine will continue to expand and provide a greater understanding of the molecular basis of disease, as well as the clinical screening and management human diseases.